Aging exacerbates the brain inflammatory micro-environment contributing to α-synuclein pathology and functional deficits in a mouse model of DLB/PD

Background Although ɑ-synuclein (ɑ-syn) spreading in age-related neurodegenerative diseases such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) has been extensively investigated, the role of aging in the manifestation of disease remains unclear. Methods We explored the role of aging and inflammation in the pathogenesis of synucleinopathies in a mouse model of DLB/PD initiated by intrastriatal injection of ɑ-syn preformed fibrils (pff). Results We found that aged mice showed more extensive accumulation of ɑ-syn in selected brain regions and behavioral deficits that were associated with greater infiltration of T cells and microgliosis. Microglial inflammatory gene expression induced by ɑ-syn-pff injection in young mice had hallmarks of aged microglia, indicating that enhanced age-associated pathologies may result from inflammatory synergy between aging and the effects of ɑ-syn aggregation. Based on the transcriptomics analysis projected from Ingenuity Pathway Analysis, we found a network that included colony stimulating factor 2 (CSF2), LPS related genes, TNFɑ and poly rl:rC-RNA as common regulators. Conclusions We propose that aging related inflammation (eg: CSF2) influences outcomes of pathological spreading of ɑ-syn and suggest that targeting neuro-immune responses might be important in developing treatments for DLB/PD. Supplementary Information The online version contains supplementary material available at 10.1186/s13024-022-00564-6.

Aging is associated with deficits in proteostasis, immune surveillance and stem cell regeneration and increased DNA damage and methylation, mitochondrial dysfunction and formation of senescent cells [10][11][12][13]. It is hypothesized that while alterations in protein homeostasis during aging might be associated with progressive accumulation of Aβ, tau and ɑ-syn [14], the other hallmark pathways of aging such as neuroinflammation and cell senescence might interact with protein aggregation to lead to neurodegeneration [13]. Under physiological conditions ɑ-syn is an intracellular protein that might play a role in neuroplasticity [15], however during aging and under pathological conditions ɑ-syn aggregates can be released to the extracellular space leading to cell to cell propagation [16,17] spreading and seeding of small aggregates into preformed protofibrils (pff ) [18,19] and fibrils in neighboring neuronal and non-neuronal cells [20]. Recent evidence has shown that the intrinsic structure of ɑ-syn fibrils dictates the characteristic of the synucleinopathies [21] and for instance inoculation of selected ɑ-syn pff into the CNS can reproduce several aspects of the pathology of DLB/PD in wild type animals models [19,21].
Although protein aggregation and spreading have been extensively studied, less is known about the contribution of aging. One possibility by which aging might lead to neurodegeneration is dysregulation in immune cell function [22][23][24][25]. This might be in part mediated by extracellular ɑ-syn propagating to glial cells [26][27][28]. For example, it has been shown that ɑ-syn can activate innate immune responses via Toll like receptors [26,[29][30][31][32][33]. Previous studies have also shown that in DLB/ PD [34][35][36] as well as in animal models [36,37] ɑ-syn might trigger inflammatory responses involving T cells. Mechanistically, a recent study proposed that an axis involving CXCR4-CCL12 signaling involving pathological interleukin-17 producing T cells might play an important role in DLB/PD pathogenesis [38].
In this study we evaluated the role of aging in neurodegeneration in the ɑ-syn pff model. We found that inoculation of ɑ-syn pff in aged mice resulted in greater spreading and deficits compared to young mice, with ɑ-syn pff-inducing gene networks in young mice that overlapped with genes differentially expressed in aged mice. Remarkably, transcriptomics analysis projected from Ingenuity Pathway Analysis uncovered a novel network that involves the colony stimulating factor 2 (CSF2)-CSF2 receptor (CSF2R) pathway as common regulator. We propose that such changes in inflammatory gene expression underlies the increased susceptibility of aged mice to enhanced ɑ-syn induced pathology and might represent a new avenue for therapeutics.

Animals
A total of 111 male and female C57BL/6 wild type mice were used in this study. The "young" group were purchased from Charles River at the age of 3-4 months old (n = 31), and the "aged" group were provided by the NIA funded rodent colonies or Charles River at the age of 18-19 months old (cohort 1, n = 35; cohort 2, n = 37). For comparison of ɑ-syn monomer and pff, an additional 8 young mice were used (Supplementary Table 1). All experiments were performed in accordance with the ALAC-and ACUC approved protocols of NIA/NIH.

Stereotaxic surgery and study design
After deeply anesthetizing mice with isoflurane, the animals were immobilized in a stereotaxic frame and bilateral stereotaxic injections were made using predetermined coordinates in the striatum (bregma, 0.2 mm; lateral, 2 mm; and depth, 3.2 mm) with a 30-gauge Hamilton syringe under aseptic conditions. All animals were observed during and after surgery, and pain killer was administered for 3 days including surgery day. Half of the mice in each group were injected 5 μg (2.5 μl of 2 μg/μl concentration) of murine ɑ-syn pff (generous gift from Dr. Kelvin Luk at University of Pennsylvania, CNDR) [39] and the other half were injected with 2.5 μl of PBS as a control. ɑ-syn pff preparations were diluted in sterile PBS to the final concentration of 2 μg/μl, stored at -80 °C freezer until use, and sonicated briefly before intracranial injection. Mice were analyzed at 1-or 3-months after the injection with attention to sex and age group balance. The experiment in the aged mice was repeated with an additional set of mice (n = 37). Additional control experiments were performed by injecting monomeric α-syn, which was prepared 1 h before the injection. For this purpose, 1 mg of lyophilized endotoxin-free human recombinant α-syn was dissolved in PBS and filtered with 0.2 µm syringe filter to remove aggregates, the monomeric α-syn was stored at -80 °C until use. The amount of monomer was determined by BCA assay and diluted into PBS, the preparation quality was tested by western blot and thioflavin T assays. For these experiments monomeric ɑ-syn and ɑ-syn pff, we injected 5 μg of ɑ-syn monomer or pff (2.5 μl of 2 μg/μl concentration) bilaterally in the striatum of 5 month old mice (n = 4 each group, total n = 8) and analyzed at 1 month after the injection by immunohistochemical analysis with the right brain hemisphere and RT-PCR analysis following microglia extraction with the left hemisphere.

Behavioral testing Open field test
Mice were placed in a 40 cm × 40 cm × 40 cm white plexiglass chamber (Maze Engineers; Boston, MA) under dim lighting and recorded for 15 min. Behavior was analyzed with ANY-Maze software (Stoelting; Wood Dale, IL). Behavior from the first 3 min was found to be most affected by ɑ-syn pff injection, so all analysis was conducted during this time period.

Fear conditioning
Mice were placed in a rectangular conditioning chamber (Med Associates) with metal bars for a floor and lit by dim white light. After a 2-min acclimation period, mice were presented with a 30-s tone that co-terminated with a 2-s, 0.5 mA scrambled shock with alternating current. A second tone-shock pairing was delivered 60 s later, and mice remained in the chamber an additional two minutes. The following day, mice were placed in the chamber for 5 min to assess freezing conditioned to the context. On day 3 the chamber was altered with insertion of a smooth plastic floor, black triangular wall inset, and illumination only by infrared lighting. Mint odor (McCormick; Hunt Valley, MD) was applied to the ceiling of the chamber to further differentiate it from the previous context. Mice were placed in the modified chamber for a 2-min baseline, and then a constant tone was played for 3 min. All freezing was recorded with a side-mounted camera and scored using automated software with default settings for freezing thresholds.

Wire hang
Mice were placed on a wire cage-top that was inverted to allow mice to hang upside down while grasping all four limbs. Latency to fall was recorded up to a maximum of 600 s. Three trials were conducted with at least 30 min between trials. Log 10 transformation on fall latencies was required to produce a normal distribution.

Rotarod
Mice were tested in eight trials on a 3.2 cm diameter drum (Med-Associates; St Albans, VT) that accelerated from 4 to 40 rpm over 300 s. On the first two days mice were tested in one and then two trials, respectively. On these days latency until the first fall was recorded, but mice were replaced on the rod after the first trial. On the third day mice underwent five consecutive trials in which they were removed from the apparatus after the first fall. Each trial was separated by at least 30 min. Latency to fall (max 300 s) was analyzed; mice approached maximal performance in the last 3 trials; therefore, averages from all trials preceding those were averaged into a single value.

Elevated zero maze
Mice were tested for five minutes in a zero-maze apparatus elevated 61 cm above the floor. The floor of the apparatus was a gray plexiglass 5 cm-wide circular track with 50 cm outer diameter. Two opposite quadrants had 20 cm tall black walls, and the quadrants in between had a 0.5 cm tall lip to contain mice. Video was analyzed with Noldus Ethovision (Wageningen, Netherlands).

Horizontal beam
On the first day mice were trained to run down a series of 1-m-long rectangular plexiglass beams (24,12, and 6 mm in diameter) towards a 20 × 20 x 20 cm dark goal box. The room was brightly lit, and beams were suspended 50 cm above the floor with a vinyl net to catch any mice that might fall. On the second day trials were recorded with an overhead camera as mice ran two trials each on the 12 and 6 mm beams. Speed in the middle 60 cm of the beam was recorded and analyzed with ANY-Maze software. Data from trials on same beam widths were averaged.

Motor impairment score
"Motor impairment score" was derived from speed on the 12 mm-wide horizontal beam, rotarod, and wire hang. Similar approaches have been used to combine data from multiple behavioral tests into a single aggregate score reflecting effects of Alzheimer's transgenes [40] or chronic stress [41]. Numbers for each individual test were first standardized into number of standard deviations above or below the age-and sex-matched control PBS group. Resulting Z-scores were averaged and multiplied by -1, such that positive scores in experimental groups indicate impaired performance. Motor impairment scores for control groups were by design 0.

Primary microglia preparation
Primary microglia were isolated from mouse brains utilizing an Adult Brain Dissociation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instruction. Briefly, enzyme-mixed brains were dissociated by gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) for 30 min. After filtration with a strainer, the brain homogenates were incubated with CD11b magnetic beads (Miltenyi Biotec) for 15 min. The incubated beads were rinsed 3 times with MACS buffer (Miltenyi Biotec) before eluting the cells.
Consistent with a recent study [42] using a similar model of synucleinopathy to ours, only about 2% or less monocyte/macrophage infiltration in the mice brains was detected. In addition to this, we performed perfusion before sacrificing the mice to minimize white blood cell contamination.

RNA extraction and bulk RNA-Seq library preparation
RNA was extracted from the microglia using RNeasy micro kit (Qiagen) as per manufacturer's instructions. RNA quality was assessed by using Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer according to the manufacturer's instructions. Qualified total RNA (RIN > 8, 10 ng) from each sample was processed by following SMARTer ® Stranded Total RNA-Seq Kit v2-Pico Input Mammalian protocol (Takara Bio USA, Inc., CA). Briefly, total RNA was converted to cDNA followed by addition of barcodes and adapters. The library was purified using AMPure beads. Takara Bio's SMART (Switching Mechanism at the 5' end of RNA Template) technology was used to remove ribosomal cDNA. After final library PCR amplification and cleaning, the libraries were quantified on the Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA Kit. The libraries were the sequenced at single end with read length of 75 bp on Illumina HiSeq 4000 platform at the Johns Hopkins Sequencing Core facility (Baltimore, MD) to yield an average depth of 70 million reads per sample.

Bulk RNA-Seq data analysis
RNA-Seq 75 bp single-end reads were analyzed with kallisto-v0.46.2, using mm10; Ensembl v96 annotation. From the Bioanalyzer results, we calculated an average fragment size of 250 bp after subtracting 120 bp for the adapter size and mean standard deviation ~ 80 [kallisto parameters for single-end: -l 250 -s 80]. We aggregated to gene level by applying tximport to kallisto TPM counts and identified covariates to exclude from the analysis by principal component analysis (R package prcomp). From the top 1000 most variable genes of age principal component (PC1), we identified the most variable genes with aging by applying elastic net regression (glmnet). Differential expression analysis for studying age effect and pff effect respectively was performed with DESeq2; and applied Benjamini Hochberg multiple test correction. We identified up-and down-regulated genes with at least twofold change at p-value ≤ 0.05. All overlap significance p-values were calculated by hypergeometric tests. Ingenuity Pathway Analysis (IPA) was used to identify enriched canonical pathways, upstream regulators and enriched functions and diseases.

Quantitative RT-PCR data analysis
For cDNA synthesis SuperScript ™ IV First-Strand Synthesis (Thermo Fisher Scientific) was used. Purified 50 ng RNA from isolated microglia was annealed to 2.5 ng/µl random hexamers and incubated with 0.5 mM dNTP mix, 5 mM DTT, 2.0U/ul RNAse Inhibitor, 2.0U/µl RNAse H, and DEPC treated water in a final volume of 40ul (as per manufacturer's instructions). 2.5 ng of cDNA was then incubated with 1 × iTaq Universal SYBR Green Supermix (BioRad), 500 nmol of each primer (Supplementary Table 2) and RNAse free water in a final volume of 15 µl. Quantitative RT-PCR was used confirm target gene expression using the following run method: 93° for 10 min; then 40 cycles of 95° for 15 s, 62° for 1 min, 95° for 15 s, and 62° for 1 min; and finally 95° for 30 min, then 62° for 15 s. Analysis was performed by ABI Prism 7500 Sequence Detection System (Applied Biosystems). Primer pairs were designed using the online Integrated DNA Technologies tool (https:// www. idtdna. com/) along with Primer v3 and NCBI Primer-BLAST primer designing softwares taking into consideration the GC%, Tm and amplicon size of 70-150 bp. The sequences of primers used are shown below in Supplementary Table 2.

Neuropathology, immunohistochemistry, and image analysis
At one-or three-months post-injection, all mice were deeply anesthetized and transcardially perfused with 30 ml of PBS. A hemisphere from each harvested brain was first fixed in 70% Ethanol in PBS and then embedded in paraffin. Then 6 µm-thick sections were cut through the entire CNS for conventional neuropathological examination and immunohistochemistry (IHC). Sections were incubated overnight at 4 °C with the following primary antibodies: 81A (anti-ɑ-syn, p-S129, gift from Drs. Virginia Lee and John Trojanowski at University of Pennsylvania, CNDR, mouse, 1:20 K, citrate buffer treatment), CD3 (abcam ab16669, or Thermo Fisher Scientific MA5-14,524 rabbit polyclonal, 1:200, citrate buffer treatment), CD4 (abcam ab183685, rabbit polyclonal, 1:1000, tris buffer treatment), GFAP (Millipore MAB3402, mouse monoclonal, 1:200) and Iba1 (microglia marker, abcam AB178846, rabbit polyclonal, citrate buffer treatment), CD8 (Cell signaling 98941S, rabbit polyclonal, 1:1000, tris buffer treatment), NeuN (Millipore MAB377, mouse monoclonal, 1:1000), tyrosine hydroxylase (Millipore AB152, rabbit polyclonal, 1:1000), CXCR4 (Cell signaling D4Z7W, rabbit polyclonal, 1:500, tris buffer treatment), MMP8 (Thermo Fisher Scientific PA5-79,687, rabbit polyclonal, 1:500 citrate buffer treatment), ADAM8 (Thermo Fisher Scientific PA5-98,303, rabbit polyclonal, 1:250), Lipocalin-2 (LCN2, abcam ab63929, rabbit polyclonal, 1:500). Sections were then incubated in biotin-tagged antimouse or anti-rabbit IgG1 (1:400, Vector Lab) secondary antibodies, followed by Avidin DHRP (1:200, ABC Elite, Vector Lab), visualized with diaminobenzidine (DAB, Vector Lab), counterstained with hematoxylin, cover slipped and imaged with a Zeiss wide field microscope. An additional set of sections were pre-treated with proteinase K (Viagen #501-PK, 10 μg/ml, 1 min) as previously described [43] and immunostained with antibodies against pS129-ɑ-syn (81A) and conformation changed ɑ-syn (syn506, gift from Drs. Virginia Lee and John Trojanowski at University of Pennsylvania, CNDR, mouse, 1:20 K, citrate buffer treatment) and visualized with DAB. For double immunofluorescence, brain sections were incubated with a combination of antibodies against p-ɑ-syn, p-S129 and T cell marker (81A/CD3) or p-S129 and Iba1 marker (81A/Iba1). Sections were labeled with FITC and Texas-red conjugated secondary antibodies, nuclei were stained with DAPI (Hoechst 33,258, Thermo Fisher H3569, 1:100,000), background was reduced with TrueBlackTM Lipofuscin Autofluorescence Quencher (Biotium: 23,007) diluted 1/40 in 70% EtOH, and tissue was mounted under glass coverslips with anti-fading media (Pro-Long ™ Gold Antifade Mountant, Thermo Fisher Scientific). Briefly as previously described [36], all sections were processed and imaged under the same standardized conditions and blind coded. Four fields from the frontal cortex, amygdala and striatum were examined for each section and performed in duplicate for each mouse. Sections visualized with DAB were imaged with an Olympus BX41 microscope and analyzed with the Image J program (NIH) to determine the number of CD3 + , CD4 + , CD8 + , Iba1 + and GFAP + cells per field (230 μm × 184 μm). As previously described stereology analysis utilizing a modified version of the disector method was used to estimate the numbers of NeuN (neocortex, striatum) and TH positive neurons (S. Nigra pars compacta) [44], while TH immunoreactivity in the striatum was analyzed by optical density [45]. Representative maps displaying the distribution of pS129-ɑ-syn (81A) pathology in the young and aged mice were achieved by digitally overlaying the low power (2x) images of the scanned immunostained brain sections into the corresponding coronal brain levels of the Paxinos and Franklin mouse brain atlas [46] and then marking them manually. For estimation of microglia cell branching, briefly as previously described [47] paraffin sections immunostained with the Iba1 antibody without counterstain were scanned at high power (900X) with an Olympus BX41 microscope and digital images of individual microglia were manually inverted, gray scaled and analyzed with the skeletonize functionality of Image J program (NIH) using a modified version of the method by Young and Morrison [48]. Double immunolabeled sections were imaged with an Apotome II mounted in a Carl Zeiss AxioImager Z1 microscope. Optical Sects. (0.5 μm thick) were analyzed via the Zen 2.3 platform to determine the average number of CD3 cells in close proximity to ⍺-syn + neuritic process.

Statistical analysis
Values shown in the figures are presented as mean ± SEM. Behavioral results were initially analyzed by analysis of variance (ANOVA) of a linear model with interacting terms for age, sex, ɑ-syn pff, and interval, with body weight (measured at beginning of study) as a covariate and an additional factor to account for batch variability. Due to the extensive number of terms in these models, only the most pertinent results are reported. Dunnett's post-hoc tests were used to compare ɑ-syn pff-injected mice vs relevant PBS-injected controls. For neuropathological analysis, P values for determination of statistical significance of the differences were calculated with one-way ANOVA with Turkey's post hoc test or unpaired Student's t test.

Inoculation of ɑ-syn pff in aged mice resulted in enhanced ɑ-syn accumulation and associated neurodegenerative deficits compared to young mice
Previous studies have shown that ɑ-syn pff injection induces neuropathological features associated with DLB/PD [18,19]. To investigate the effects of aging on the ɑ-syn neuropathology and related deficits, first we injected ɑ-syn pff into the striatum of a young mouse cohort and examined ɑ-syn distribution and dissemination at 1-and 3-months after injection. As expected, the control (PBS) injected mice did not show the characteristic ɑ-syn pathology in any brain region when immunostained with the antibody against phosphorylated ɑ-syn (p-ɑ-syn) ( Fig. 1A, C, E, G, left panels). In contrast, the ɑ-syn pff injected young mice showed Lewy body like p-ɑ-syn pathology mainly in the neocortex and amygdala and Lewy neurite like p-ɑ-syn positive pathology in the neuropil in the striatum at 1-month after injection ( Fig. 1A). At 3-months after injection, we observed more robust p-ɑ-syn pathology in all neocortices, amygdala and striatum (Fig. 1C). Image analysis of p-ɑ-syn percentage area of neuropil showed significant increase of p-ɑ-syn in the ɑ-syn pff injected young mice at 1-and 3-months after injection compared to the PBS injected young mice (Fig. 1B, D). More detailed representation of the topographical distribution of the p-ɑ-syn immunoreactive aggregates throughout the brain of the young mice at 1-and 3-months post injection is illustrated in Figure  S1A.
Next, we injected ɑ-syn pff into the striatum of 18-19 month old mice to test if aging affects transmission of ɑ-syn pathology. We analyzed the brains at 1and 3-months after injection. Intracranial injection of ɑ-syn pff into aged mice induced robust p-ɑ-syn pathology in the neocortex, amygdala, and striatum at 1 month ( Fig. 1E) and 3 months post injection (Fig. 1G). Image analysis of p-ɑ-syn percentage area of neuropil showed significant increase of p-ɑ-syn in the ɑ-syn pff injected aged mice at 1-and 3-months after injection compared to the PBS injected aged mice in all brain regions (Fig. 1F, H). Comparison of p-ɑ-syn percentage area of neuropil between young and aged mouse cohorts revealed that the aged mouse cohort showed higher percentage of p-ɑsyn neuropil in amygdala at 1-and 3-months and striatum at 1 month after injection. The neocortex showed a Fig. 1 Phosphorylated-ɑ-syn immunostaining in young and aged mice with PBS or ɑ-syn pff injection. A Representative images of p-ɑ-syn immunostaining in young mouse cohort at 1-month post injection. Images are from three different mice (1, 2 and 3) of three brain regions (M1/M2 or somatosensory cortex (Neocortex), basolateral/basomedial amygdala (Amygdala), and dorsal striatum (Striatum)). Left panels are PBS (vehicle) injected and right are ɑ-syn pff injected. B Image analysis of p-ɑ-syn % area of neuropil of young mouse cohort at 1-month post injection. C p-ɑ-syn immunostaining of young mouse cohort at 3-months post injection. Same format as (A). D Image analysis of p-ɑ-syn % area of neuropil of young mouse cohort at 3-months post injection. E p-ɑ-syn immunostaining of aged mouse cohort at 1-month post injection. Same format as (A). F Image analysis of p-ɑ-syn % area of neuropil of aged mouse cohort at 1-month post injection. G p-ɑ-syn immunostaining of aged mouse cohort at 3-months post injection. Same format as (A). H Image analysis of p-ɑ-syn % area of neuropil of aged mouse cohort at 3-months post injection. I-K Comparison of image analysis of p-ɑ-syn positive % area of neuropil in neocortex (I), amygdala (J) and striatum (K) of ɑ-syn pff injected mice at 1-and 3-months post-injection in young (blue) and aged (red) mouse cohorts. Scale bars, 25 μm. Data are mean ± SEM. Unpaired t test was used. ***p < 0.001; ****p < 0.0001 tendency for higher percentage after 3 months post injection, but differences between young and aged were not significant ( Fig. 1I-K).
Consistent with the higher power image analysis of the p-ɑ-syn aggregates in the selected brain regions, distribution maps of the p-ɑ-syn aggregates showed that in the aged animals there was a more abundant presence of aggregates in neocortical regions (orbital, prelimbic, anterior cingulate, primary somatosensory, primary motor, retrosplenial, temporal association, entorhinal, visual cortex and posterior parietal association), amygdala, striatum, and substantia nigra pars compacta, while the hippocampus and thalamus and other subcortical nuclei displayed few or no p-ɑ-syn inclusions (Fig.  S1B). The p-ɑ-syn aggregates in the substantia nigra were somewhat different to those in the neocortex in that they were more discrete, granular or finely fibrillar in appearance and extended from the perikaryal into the neuritic processes with relative more abundance in aged mice ( Fig. S2A, B). To further characterize the ɑ-syn aggregates in the ɑ-syn pff injected mice, selected sections were treated with proteinase K (PK) before immunostaining with antibodies against p-ɑ-syn (81A) and misfolded conformation changed ɑ-syn (syn506). Staining with the 81A antibody without PK pretreatment revealed discrete fibrillar inclusions that were more abundant in aged mice (Fig. S3A). Pretreatment with PK resulted in a considerable accentuation of the pathological fibrillar inclusions in both young and aged mice, suggesting that they are composed of mostly insoluble p-ɑ-syn aggregates (Fig. S3A). In contrast, with the syn506 antibody (without PK) there was intense immunostaining of the neuropil with a punctate pattern as well as inclusions in the neuronal bodies and neuritic processes in young mice, all of which were more abundant in aged mice (Fig. S3B). Pretreatment with PK considerably decreased the neuropil immunolabeling and enhanced the neuronal and neuritic fibrillar inclusion patterns in young and aged mice (Fig. S3B), suggesting that the ɑ-syn in the neuropil is mostly soluble native protein, whereas inclusions consist of misfolded pathological ɑ-syn.
To account for inter-mouse variability in freezing behavior, we examined the ratio of freezing to cue: context. Here the main effect of ɑ-syn pff was more pronounced (F(1,84) = 7.55, p = 0.0070), and there was a marginally significant ɑ-syn pff x age x sex interaction (F1,84 = 3.91, p = 0.051). ɑ-syn pff significantly reduced this ratio at 3 months post-injection in aged females (p < 0.05) and in all aged mice combined (p < 0.01) (Fig. 2D). There were no significant main or interacting effects of ɑ-syn pff on freezing during training baseline (Fig. S4A) or on contextual testing day (Fig. S4B).
Performance in the wire hang test was impaired following ɑ-syn pff injection (ɑ-syn pff main effect F1,84 = 6.93, p = 0.010), with an ɑ-syn pff x sex interaction (F1,84 = 4.59, p = 0.035). To combine data within sexes across different ages, which were highly different (F1,84 = 119.44, p = 1e-17), we normalized each mouse's measurement to percent of age/sex/batch matched controls. Within all combined male mice, there was a progressive decline in performance with significant differences in the ɑ-syn pff group when compared to PBS controls at 1 month (p < 0.05) and 3 months (p < 0.001) after ɑ-syn pff injection (Fig. S4C). Within the combined female population there was no difference between PBS and ɑ-syn pff injected mice (Fig. S4D). In rotarod, there was a main effect of ɑ-syn (F(1,84) = 8.45, p = 0.0097). However, when post-hoc comparisons were made within age and sex groups, there were no differences between PBS and ɑ-syn pff injected mice. These results suggest marginal influence of ɑ-syn pff injection at the post-surgical time points evaluated here (Fig. S4E, F). There were no significant main or interacting effects of ɑ-syn pff on speed on the 12 mm or 6 mm beam (Fig. S4G, H). Motor impairment score, which indicates combined impairments in rotarod, wire hang, and horizontal beam tests, showed significant main effects of ɑ-syn pff (F1,84 = 5.81, p = 0.018) and ɑ-syn pff x sex interaction (F1,84 = 4.48, p = 0.037). Post-hoc tests revealed that this score was Iba  open field test in the aged mouse cohorts separated by sex. C Percentage of freezing time to the cue in the fear conditioning test. D The ratio of freezing to cue:context in the fear conditioning test. E Combined scores from three locomotor tests (wire hang, rota-rod and horizontal beam). F Each test was normalized by calculating how many standard deviations worse than age/sex-matched PBS controls. Data are mean ± SEM. One-way ANOVA with Dunnett's post-hoc tests were used. *p < 0.05; **p < 0.01 significantly impaired in aged male mice at 3 months post-pff injection (p < 0.05) (Fig. 2E, F). To internally validate the motor impairment score, we assessed its sensitivity in detecting aging-induced impairment. We calculated this measure using only PBS-injected mice, with young animals designated as controls. Indeed, resulting scores in aged male and female mice were substantially impaired (main effect of age group F1,42 = 75.83, p = 6e-11), confirming that our method of calculating motor impairment is highly sensitive to detecting decline in motor function.
Given the behavioral deficits we next analyzed if aging in combination with ɑ-syn pff promoted neurodegenerative pathology. For this purpose, brains were evaluated with the pan-neuronal marker NeuN and the dopaminergic marker TH. Immunohistochemical and image analysis of neuronal populations in the neocortex showed comparable numbers of neurons in the young mice with no difference between PBS and ɑ-syn pff injected mice at 1-and 3-months post injection (Fig. S5A, B). However, in the aged mice, there was an approximate 25% loss of neocortical neurons in the ɑ-syn pff injected mice at 1and 3-months post injection (Fig. S5A, B). In the striatum there was an approximate 20% loss of neurons in the young (at 1 but not 3 months) and 25% in the aged ɑ-syn pff injected mice at 1-and 3-months post injection (Fig.  S5C, D). Likewise, levels of TH immunoreactive fibers in the striatum were reduced between 30-40% in the young and aged ɑ-syn pff injected mice at 1-and 3-months post injection (Fig. S6A, B), suggesting a loss of dopaminergic axons in the striatum of ɑ-syn pff treated mice that was enhanced with aging. No differences were observed in the numbers of TH positive cells in the S. Nigra between PBS and ɑ-syn pff injected young and aged mice at 1-and 3-months post injection (Fig. S6C, D).
Together, aging increased ɑ-syn pff associated pathology in the neocortex, limbic system and striatum resulting in more pronounced memory deficits as reflected by the fear conditioning test and neurodegenerative pathology.

Enhanced infiltration of adaptive immune cells in aged mice inoculated with ɑ-syn pff compared to young mice
We and others have shown [36,37] inflammatory responses involving T cells in mouse models of DLB/ PD. To test the effects of aging on T cell infiltration in the ɑ-syn pff model, we first performed immunohistochemistry with anti-CD3 antibody (T cell marker). We observed very few CD3 + cells in PBS-injected control mice of any age-group (Fig. 3A, C, E, G, left panels). However, young mice injected with ɑ-syn pff showed a considerable infiltration by CD3 + cells in the neocortex, amygdala, and striatum at 1 month after injection (Fig. 3A, right panel), that decreased at 3 months after injection (Fig. 3C, right  panel). These T cells were observed around blood vessels and in the interstitial space in the neuropil. Image analysis of CD3 + cell counts showed significant increase of CD3 + cells in neocortex, amygdala, and striatum at 1 month post injection, and neocortex and striatum at 3 months post injection (Fig. 3B, D). In contrast, aged mice that were injected with ɑ-syn pff showed considerably more robust infiltration by CD3 + cells at 1 month after injection (Fig. 3E, right panel). Although the frequency of CD3 + cells was reduced at 3 months after injection (Fig. 3G, right panel), the numbers remained above those seen in young mice. Image analysis of T cells showed significant increase in the ɑ-syn pff injected cohort compared to PBS injected controls in all brain regions at both 1-and 3-months post injection (Fig. 3F,  H). Comparison of CD3 + cell counts between young and aged mouse cohorts revealed that aged mice showed significantly higher CD3 + cell counts in the neocortex, amygdala, and striatum at both 1-and 3-months after injection of ɑ-syn pff (Fig. 3I-K). Likewise, in the substantia nigra of the young and aged PBS injected cohorts there were rare CD3 + cells that were relatively more abundant in the ɑ-syn pff mice (Fig. S2C, D).
To identify subsets of CD3 + cells, we stained slides with antibodies against either CD4 or CD8. While the PBS injected young and aged mice displayed only a few CD4 + cells after injection (Fig. S7A, C, E, G, left panels), remarkably, the ɑ-syn pff injected young and aged mice at 1-month after injection showed several CD4 + cells in neocortex, amygdala and especially striatum (Fig.  S7A, E, left panels), and these decreased at 3-months after injection (Fig. S7C, G, left panels). Image analysis of CD4 + cell counts between PBS and ɑ-syn pff injected mice showed significantly increased CD4 + cells in ɑ-syn pff injected cohorts within all brain regions inspected in both young and aged cohorts at 1-and 3-months after injection, with the exception of amygdala and striatum tissue of young mice at 3-months after injection (Fig.  S7B, D, F, H). Comparison of CD4 + cell counts between young and aged mice cohorts at 1-and 3-months post injection showed greater CD4 + cells infiltration in the aged mice cohort, more prominently at 1-month after injection, with decreasing numbers 3-months after injection in both young and aged mice cohorts (Fig. S7I-K). Likewise, though only a few CD8 + cells were detected after PBS injection (Fig. S8A, C, E, G, left panels), the ɑ-syn pff injected young and aged mice at 1-month after injection showed increased numbers CD8 + cells in neocortex, amygdala and especially striatum (Fig. S8A, E, left panels), and these decreased at 3-months after injection (Fig. S8C, G, left panels). Image analysis of CD8 + cell counts between PBS and ɑ-syn pff injected mice showed significantly increased CD8 + cells in ɑ-syn pff injected cohorts of all brain regions inspected in the young mice and in the striatum and amygdala of aged cohorts at 1and 3-months after injection (Fig. S8B, D, F, H). Comparison of CD8 + cell counts between young and aged mice cohorts at 1-and 3-months post injection showed greater CD8 + cells infiltration in the aged mice cohort, more prominently at 1-month after injection, with decreasing numbers 3-months after injection in both young and aged mice cohorts (Fig. S8I-K).
To further investigate that the effect on T cells were selective to ɑ-syn pff and not a non-specific effect of the  2 in neocortex (I) amygdala (J) and striatum (K) of ɑ-syn pff injected mice at 1-and 3-months post-injection in young (blue) and aged (red) mouse cohorts. Scale bars, 40 μm. Data are mean ± SEM. Unpaired t test was used. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 ɑ-syn preparation, we compared the effects of injecting the ɑ-syn monomers vs pff in a subset of comparable young wild-type mice and analyzed for T cells and inflammation after 1 month. As expected, compared to pff, ɑ-syn monomers did not trigger accumulation or spreading of ɑ-syn from the striatum to the neocortex and amygdala like PBS injection (Fig. 4A, B). Moreover, like mice injected with PBS, animals that received ɑ-syn monomer injection only showed scattered infiltration by CD3+, CD4+, and CD8+ T cells (Fig. 4C-E). In contrast, mice that received ɑ-syn pff injection showed increased numbers of CD3+, CD4+, and CD8+ T cells mostly in the striatum and amygdala (and to a lesser extent in the neocortex) (Fig. 4C-E). Together, these results show that aging in combination with spreading of the ɑ-syn pff pathology promotes increased T cell (CD4+ and CD8+) infiltration in the CNS of ɑ-syn pff injected mice.

Sustained presence of activated microglia in the CNS of aged mice injected with ɑ-syn pff compared to young mice
As we and others have shown that innate cells play a role in the pathogenesis of DLB/PD in ɑ-syn transgenic models [50,51], we investigated the contribution of aging on microglial and astroglial cells in the ɑ-syn pff model. Immunohistochemical analysis with the Iba1 antibody showed branched and activated microglia in neocortex, amygdala, and striatum of the ɑ-syn pff injected in both young and aged cohorts (Fig. 5A, C, E, G, right panels) compared to PBS injected mice (Fig. 5A, C, E, G, left panels). Interestingly, differences between PBS and ɑ-syn pff injected cohorts at 1 month returned to baseline at 3-months post injection (Fig. 5B, D, F, H). In contrast, comparison of Iba1 positive cells between young and aged cohorts showed greater numbers and level of activation of microglial cells in the aged mice at 1-and 3-months post injection (Fig. 5I-K). In the substantia nigra, microglial cells showed similar characteristics in the young and aged groups of mice (Fig. S9A,  B). Further analysis of process branching showed that in the young PBS injected cohorts at 1 and 3-months post injection, the microglia displayed abundant ramifications (Fig. S10A, B) while the young ɑ-syn pff injected mice showed decreased branching (Fig. S10A, B). Compared to the young mice, the aged vehicle treated mice displayed a trend toward decreased branching. Moreover, when compared to the vehicle injected aged group, the ɑ-syn pff injected aged mice displayed decreased microglia ramification at 1-and 3-months post injection (Fig. S10C, D). Taken together these results suggest that microglia in aged and mice injected with ɑ-syn pff are in an activated state. G Image analysis of GFAP optical density. Data are mean ± SEM. One-way ANOVA with Tukey's post hoc test was used. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bar, 40 μm In terms of GFAP positive astroglial cells, we observed significant astrogliosis in the neocortex, amygdala, and striatum in the ɑ-syn pff injected young and aged cohorts (Fig. S11A, C, E, G, right panels) compared to PBS injected young and aged cohorts (Fig. S11A, C, E, G, left panels) at both 1-and 3-months after injection (Fig.  S11B, D, F, H). Image analysis of GFAP positive astroglial cells between young and aged mice cohorts (PBS and ɑ-syn pff ) mostly showed no differences between GFAP positive cells at 1-and 3-months after injection, only the neocortex of ɑ-syn pff injected mice after 3 months showed increased in astrogliosis with aging ( Fig. S11I-K). In the substantia nigra compared to the PBS injected and striatum (K) of ɑ-syn pff injected mice at 1-and 3-months post-injection in young (blue) and aged (red) mouse cohorts. Scale bars, 20 μm. Data are mean ± SEM. Unpaired t test was used. **p < 0.01; ***p < 0.001; ****p < 0.0001 mice in the ɑ-syn pff injected mice showed intense astrogliosis that was enhanced with aging (Fig. S9C, D).
Control experiments where mice were injected with monomeric vs pff ɑ-syn showed that mice injected with monomeric ɑ-syn displayed similar effects to the PBS in terms of astroglial and microglial responses, while the mice injected with pff mice showed a mild increase in microglia in the striatum (Fig. 4A, F) and astrogliosis in all three regions (Fig. 4A, G). We infer that sustained astroglial and microglial activation contributes to neuropathological and behavioral deficits during ɑ-syn induced disease progression in aged mice.
To investigate the relationship between the p-ɑ-syn aggregates and the T and microglial cell infiltration, double immunolabeling studies with the antibodies against CD3 or Iba1 and p-ɑ-syn were performed in the young and aged mouse cohorts at 1-and 3-months after injection (Fig. 6A, G). In the young mice, the CD3+ cells were usually far from the ɑ-syn aggregates in the neocortex, with closer proximity in the amygdala and striatum at 1-and 3-months post injection (Fig. 6B, C). However, in Fig. 6 Double immune-fluorescence analysis in young and aged mice with ɑ-syn pff injection. A Double immunofluorescence staining with phosphorylated ɑ-syn (p-ɑ-syn, 81A) and CD3 antibodies. Split and merged representative microscopy images from the somatosensory cortex, basolateral and basomedial amygdala, and dorsal striatum of ɑ-syn pff injected aged mouse double labeled with antibodies against p-ɑ-syn (green) and CD3 cells (T cells, red channel). B, C Quantitative analysis of distances between p-ɑ-syn and CD3 cells in neocortex, amygdala, and striatum in 1-month (B) or 3-months (C) post injection. D-F Comparison of image analysis of proximity between p-ɑ-syn and CD3 cells in neocortex, amygdala, and striatum in young and aged mouse cohorts. G Double immunofluorescence staining with Iba1 and CD3 antibodies. Split and merged representative microscopy images from the neocortex, amygdala, and striatum of ɑ-syn pff injected aged mouse double labeled with antibodies against p-ɑ-syn (green) and Iba1 (microglia cells, red channel). H, I Quantitative analysis of distances between p-ɑ-syn and microglia cells in neocortex, amygdala, and striatum in 1-month (H) or 3-months (I) post injection. J-L Comparison of image analysis of proximity between p-ɑ-syn and microglia cells in neocortex, amygdala, and striatum in young and aged mouse cohorts. Scale bars, 20 μm in low magnification, 10 μm in the high magnification. Data are mean ± SEM. Unpaired t test was used. * p < 0.05; ** p < 0.01; *** p < 0.001 the aged mice there was closer proximity between p-ɑsyn neuropil threads and CD3+ cells in the amygdala and striatum at 1 month (Fig. 6B) and all three regions of the brain analyzed at 3 months (Fig. 6C). Direct comparison between young and aged mice confirmed the increased localization between T cells and p-ɑ-syn aggregates in aged mice in all 3 regions (Fig. 6D-F). Overall, compared to the T cells (Fig. 6A), the microglial cells and their processes were usually in closer proximity to the ɑ-syn aggregates (Fig. 6G). In the young mice, the microglial cells were at some proximity to the ɑ-syn aggregates in the neocortex, with closer proximity in the amygdala and striatum at 1-and 3-months post injection (Fig. 6H,  I). However, in the aged mice there was greater proximity between p-ɑ-syn aggregates and microglial cells with their processes surrounding the ɑ-syn positive cells in all brain regions at 1 month (Fig. 6H) and in the neocortex and amygdala at 3 months (Fig. 6I). Direct comparison between young and aged mice confirmed the greater proximity between microglial cells and ɑ-syn aggregates in the aged mice ( Fig. 6J-L). Taken together these studies support the notion that microglial cells might be interacting with ɑ-syn propagating aggregates during aging.

Transcriptomic analysis shows enhanced inflammation in the ɑ-syn pff injected brains in aged mice compared to young mice
To characterize the molecular link between aging and ɑ-syn pff-induced microglial activation, we assayed the transcriptomes of purified microglia isolated from PBS and ɑ-syn pff injected mice in young and aged cohorts. We found that the first principal component clearly distinguished gene expression profiles in cells from young and aged mice (Fig. S12A). We first examined the effects of age by comparing PBS injected samples using DESeq2 and adjusting for multiple testing with Benjamini-Hochberg (BH) approach (adjusted p-value < 0.1). We identified 622 genes that were up-regulated (fold change > 2) (154 ≥ fourfold genes shown for visualization purpose) and 188 genes that were down-regulated (fold change > 2) with age (p < 0.05) (Fig. 7A). That principal component (PC1) had a strong aging component was further substantiated by comparing age-associated differentially expressed genes (DEG age ) with the top 1000 PC1 genes identified by lasso regression (Fig. S12B). We found that approximately 30% of DEG age genes were in this group (gene set overlap p value 5.8e-51, including C3, Il1rn, Olr1, Rxrg, Fam71a and Plk5) (highlighted in Fig. S12B). Ingenuity Pathway Analysis (IPA) showed that DEG age were enriched for inflammatory and immune system-associated pathways like 'Communication with innate and adaptive immune cells' , ' Altered T and B cell signaling in Rheumatoid Arthritis' or pathways linked to IL-12 signaling while well-known upstream regulators of inflammation such as lipopolysaccharide (LPS), TNFɑ, IL-1b and IL-6 were also implicated (Figs. 7B, S13A). One of the upstream regulators, IL10RA which is a part of IL10 mediated anti-inflammatory cellular response, is highly repressed (Fig. 7B, right). A network constructed from pathways with more than 5 common enriched genes (or other molecules) that change with age highlighted inflammation and immune system-associated pathways like 'Communication with innate and adaptive immune cells' , ' Altered T and B cell signaling in Rheumatoid Arthritis' or pathways linked to IL-12 signaling were linked (Fig. 7C). Based on the transcriptional upstream regulators projected from IPA, a network of the top upstream regulators and their target genes (or other molecules) was constructed (Fig. 7D). Remarkably, this analysis revealed novel networks including CSF2 and poly rl:rC-RNA as well as other better known pathways such as LPS/TLR and TNFɑ as common upstream transcriptional regulators which imply that the ageassociated genes were related to inflammatory response. Cumulatively, these observations support the notion of accentuated inflammatory phenotype of microglia from aged mice. While age-associated inflammation has been reported in many mouse tissues including microglia [52][53][54], our gene set included many hitherto unknown genes (such as the CSF2/CSF2R signaling cascade) and provides the baseline for evaluating the effects of ɑ-syn pff injection. A subset of genes dysregulated by age, as observed by RNA-Seq, were validated by quantitative RT-PCR (Fig.  S13B, C).
We next identified genes that were differentially expressed in response to ɑ-syn pff injection in young and aged mice (Fig. 8A). Analyses of these gene lists revealed several interesting features. First, ɑ-syn pff induced distinct responses in microglia from young and aged mice. Gene ontology analyses of upregulated genes using Topp-Gene [55] showed that 'Regulation of immune processes' was the only pathway (amongst the top 3) that was shared between the young and aged cohorts at the 1 month post injection. Similarly, 'neurogenesis' and 'neuronal development' pathways were the only ones amongst down-regulated genes that were common to both young and aged mice, respectively, at 1 month after injection. Second, beyond the small overlap at 1 month, gene expression trajectories in young and aged microglia diverged considerably. Most strikingly, whereas responses in young mice evolved to a robust 'myeloid activation' phenotype at 3 months after ɑ-syn pff injection, those in aged mice were dominated by pathways associated with cell death ('positive regulation of cell death' and 'response to glucocorticoids') and dysregulated proteostasis ('de novo protein folding' and 'establishment of protein localization') (Figs. 8A, S14A). A subset of genes dysregulated by age or ɑ-syn pff challenge, as observed by RNA-Seq, were validated by quantitative RT-PCR (Fig. 8B). 30-50% of the genes dysregulated by 3 months of ɑ-syn pff treatment in aged mice changed in the same direction in an independent cohort of aged mice, however these trends didn't reach significance (p > 0.05) (data not shown). Genes that changed in response to ɑ-syn pff injection (≥ 1.5-fold, p ≤ 0.01) were processed through IPA to identify general features of these responses. We found that the top canonical pathways were associated with neuronal system, neuroinflammation and leucocyte signaling (highlighted in yellow) which were uniformly repressed across the four groups (Fig. S14B, left). Most upstream transcriptional regulators of DEGs behaved uniformly across all the groups (center) and similar results were found for the functions and diseases (Fig. S14B, right).
Third, a significant proportion of gene expression changes induced by ɑ-syn pff (p < 0.05) in young mice coincided with those that occurred with age (p < 0.05). Specifically, about a third of the genes that were upor down-regulated by ɑ-syn pff 1 month post injection changed in the same direction with age (Fig. 8C). At 3 months after ɑ-syn pff injection almost half the Fig. 7 Characterization of age-associated genes in microglia. A RNA from microglia of PBS injected young and aged mice were compared to identify the age-associated differentially expressed genes (DEGs). The RNA-Seq identified 622 up-and 188 downregulated genes (≥ twofold change, p < 0.05) (for visual purpose, 154 ≥ fourfold upregulated genes have been shown). For the age-associated comparison, PBS injected control mice from 1-and 3-months (including repeats) post injection groups were combined and the TpM normalized values of the DEGs are represented in the heatmap. The number of DEGs are mentioned on the left of the heatmap with few of the prominent genes mentioned on the right. B The age-associated differentially expressed genes (both up-and downregulated) were analyzed with IPA to identify enriched canonical pathways (left), and upstream regulators of the genes (right). The canonical pathways show the most enriched biological processes along with the percentage of up-(red) and downregulated (green) genes in each pathway with total number of genes mentioned outside the bar graphs. The -log p-value from the enrichment analysis is represented by a yellow dot. The upstream regulator plot identifies the cascade of upstream transcriptional regulators whose expression is affected by aging. Prominent immune regulators are noted. The bar graph shows the activation Z-score where a positive score implies activation of the transcriptional regulator while the negative score indicates repression. The color of the bar graph represents the p-value. C Based on the canonical pathways derived from IPA, a network was constructed comprising of pathways with more than 5 common enriched genes (or other molecules) that changed with age. D Based on the transcriptional upstream regulators obtained from IPA, a network of the top upstream regulators (CSF2, LPS, TNF and poly rl:rC-RNA) and their target genes (or other molecules) was constructed. This plot shows how the upstream regulator networks overlap via their target genes pff-induced genes in young mice coincided with genes that were up-regulated by age alone, suggesting that pff continued to induce an aging phenotype up to 3 months after injection in young mice. By contrast, ɑ-syn pff injection in aged mice induced age-associated genes strongly only at 1 month post injection (Fig. 8C). A subset of genes dysregulated by both age and ɑ-syn pff injection, as observed by RNA-Seq, were validated by quantitative RT-PCR (Fig. S14C). Many genes induced by ɑ-syn pff or by aging alone were classified as inflammation-associated in the MouseMine database [56] (Fig. S14D).
To further evaluate the specificity of the effects of ɑ-syn pff on gene expression, we compared the effects of ɑ-syn monomers to those of ɑ-syn pff at 1 month post intra-striatal injection. We found that mRNA levels for MMP9 and LilR4R changed in the same direction in response to ɑ-syn pff when compared to monomers or control PBS injection (Fig. S15A). Similar non-significant trends were observed for ADAM8, LCN2 and MMP8 (Fig. S15A). Finally, further confirmation of the RT-PCR results when comparing young vs aged mice injected with PBS or ɑ-syn pff (Figs. 8,   Fig. 8 Effect of ɑ-syn pff injection on microglial gene expression. A Genes dysregulated by 1-and 3-months of ɑ-syn pff injection in young and aged mice were identified by DESeq2 (fold change ≥ 2, p < 0.05). The expression levels of the DEGs are represented as TpM normalized values for young mice in the upper row and for the aged mice in the lower row. The number of DEGs are indicated to the left of the heatmap. The top 3 pathways associated with each group were determined from ToppGene database as shown below the respective heatmaps. B RNA-Seq results were validated by qRT-PCR for selected candidates. Samples were analyzed in duplicate and expression levels were normalized against Gapdh. The mean and standard error are derived from combining all samples in a group. C3, Mmp8 and Lcn2 go up with ɑ-syn pff injection while Dlk1, Kif5a, Laurp1l, Gfap2 and Pagr1a go down with ɑ-syn pff injection as also observed in RNA-Seq results. The p-value from the t-test between PBS and ɑ-syn pff data is shown above the bar graphs. C Comparison of ɑ-syn pff-induced genes with age-associated genes in microglia. Genes that were up-or downregulated in response to ɑ-syn pff (X axis) were compared with genes up-(purple bars) or downregulated (orange bars) with age. Data shown is for young and aged mice as indicated 1or 3 months after ɑ-syn pff injection. The significance of overlap was confirmed by hypergeometric test. * p < 0.05; ** p < 0.01; *** p < 0.001 S13, S14) was performed by immunohistochemistry in a subset of the genes. For example, ADAM8 immunoreactivity was mostly detected in neurons and in agreement with the RT-PCR was elevated in the young ɑ-syn pff injected mice as well as in the aged mice aged mice in the neocortex, amygdala, and striatum (Fig. S16A). Likewise, CXCR4 immunoreactivity was detected in neuronal and non-neuronal cells and increased in the young ɑ-syn pff injected mice as well as in the aged mice in the neocortex and amygdala but not the striatum (Fig. S16B). LCN2 immunoreactivity was observed surrounding the neuronal cell bodies and in axons; levels were increased in young and aged mice that were injected with ɑ-syn pff (Fig. S16C), while MMP8 was slightly increased in the amygdala of the young ɑ-syn pff and more apparent in the aged ɑ-syn pff tissue (Fig.  S16D).

Discussion
The present study showed that aging resulted in more extensive accumulation of ɑ-syn in the striatum and amygdala, greater infiltration of T cells and microgliosis Fig. 9 Schematic depiction of the potential microglial networks and pathways that interact in aging and synucleinopathies. The effects of aging processes and extracellular ɑ-syn convergence in the macrophages/microglia in the CNS probably by engaging both Toll-like receptors (TLRs) such as TLR2 and colony stimulating factor receptors (CSF-R) that in turn activate pro-inflammatory LPS-like pathways with increased production of cytokines including TNFɑ, IL6, IL10ɑ and activation of networks involving CSF2. This in turn results in a cascade of events involving further activation of microglia, astrocytes and trafficking of T cells into the CNS parenchyma that jointly produce pro-inflammatory neurotoxic factors leading to neurodegeneration and behavioral deficits. Distinctive inflammatory transcriptomic patterns in microglia showed that ɑ-syn pffinduced gene networks in young mice (eg: LPS, TNFɑ, IL1b, IL6) overlapped with genes differentially expressed in microglia of aged mice (Fig. 9). Moreover, based on the IPA, a network including novel targets such as CSF2 and poly rl:rC-RNA were identified as top upstream regulators. We infer that the persistent ɑ-syn pff challenge initially induces an aging phenotype that is followed by a distinct and presumably more deleterious gene expression program. This transition occurs between 1 and 3 months after ɑ-syn pff injection in aged mice but not for at least 3 months after ɑ-syn pff injection in young mice. Interestingly, ɑ-syn pff did not up-regulate ageassociated genes in aged mice even at the 1-month post injection, suggesting that aging had already conferred an early ɑ-syn pff-like phenotype with respect to this subset of genes.
These results are consistent with the concept that ɑ-syn aggregates might lead to neurodegeneration by dysregulating adaptive and innate immune responses [22][23][24]57] and that aging plays an important role in this process. For example, a recent study showed in a similar model of synucleinopathy that blocking A1 astrocyte conversion by microglia using glucagon-like peptide-1 receptor (GLP1R) agonists is neuroprotective, supporting a role of the innate immune system [58]. Moreover, other studies have shown that intra-striatal injection of ɑ-syn pff results in activation of microglia and astrocytes, as well as infiltration of B, CD4 + T, CD8 + T, and natural killer cells in the brain at 5 months post injection. In one such study, the authors showed alterations in the frequency and number of lymphoid cell subsets in the spleen and lymph nodes with minimal alterations in the blood [42]. A similar study showed in 2-and 5-month old mice that ɑ-syn pff triggered dopaminergic deficits only in the 5 month old mice [59]. In addition, a study in Sprague Dawley rats demonstrated that intra-striatal injection of ɑ-syn pff triggered MHCII-expressing cells that are composed of both resident microglia as well as cells from the periphery such as monocytes/macrophages, and T cells [60]. Our study is different in that while previous studies were conducted in young (2-3 months old) or adult (5 months old) rodents with analysis performed in mice at 5 months post injection and in rats after 1, 3 and 6 months post injection, in the present study the focus was to investigate the mechanisms and effects of aging (18-19 months old) on immune responses mediated by extracellular ɑ-syn. Interestingly, in support of our findings a recent study showed that inoculation of ɑ-syn fibrils in the gut of aged (but not in the young) mice resulted in ɑ-syn pathology in the midbrain accompanied by motor deficits [61].
The motoric deficits observed in the ɑ-syn pff injected aged mice might be related to the accumulation of ɑ-syn in the striatonigral system with dopaminergic fiber deficits. Here it is possible that the propagation of ɑ-syn pathology in the motoric and somatosensory cortex might also contribute to the deficits. Although in agreement with a previous study [62], we did not observe neuronal loss in the neocortex of the young ɑ-syn pff injected mice, the aged mice displayed subtle neurogenerative pathology in the motor and somatosensory cortices that might contribute to the composite motor alterations, we found in the aged mice. Also, in accordance with the study by Stoyka et al. [62] we found that the aged ɑ-syn pff injected mice displayed non-motor alterations namely fear conditioning deficits that might be potentially explained by the age dependent accumulation of ɑ-syn and inflammatory insults in the amygdala. While our study showed primarily loss of TH-positive dopaminergic fibers in the striatum of the young and aged mice injected with ɑ-syn pff, we did not detect a significant loss of TH neurons as other studies have shown [63]. Given that the focus of our study was on neuroinflammation and aging, our analysis in young and aged mice was done only after 1-and 3-months post injection; in contrast, most studies showing TH cell loss required 6 months or longer after injection to do so [19,63]. Humans with familial PD LRRK2 mutations show evidence of hyperdopaminergic function in PET scans performed at the pre-symptomatic stage [64]; similarly, increased extracellular dopamine is seen in ɑ-syn overexpressing mice at ages that precede dopamine loss and locomotor deficits [65]. Accordingly, transient hyperactivity is seen at early stages that precede dopamine neuron loss and behavioral deficits in fine motor control in both ɑ-syn overexpression and ɑ-syn pff models [19,66,67]. It is not well understood the degree to which early changes such as these simply to delay onset of symptoms, or if they play a causal role in the clinical progression of disease pathology and symptoms [68]. Interestingly, we observed dissociation between the early hyperactivity and later motor deficits following ɑ-syn pff injection. In aged mice, we saw hyperactivity in females but not in males; conversely, motor impairments were seen in males but not in females. Furthermore, aging caused a delay in the postinjection hyperactivity but acceleration in development of motor deficits. Our findings suggest the possibility that early increases in dopamine function may be mechanistically unrelated to later dopaminergic neuron loss. Beyond locomotor effects, we also found cognitive deficits following ɑ-syn pff administration. Unlike the motor impairments, however, there were no sex differences in the cognitive impairment among aged mice. Thus, aging differentially accelerates motor and cognitive impairments caused by ɑ-syn pff seeding, in a sex-dependent manner.
In terms of the specificity of the effects of the ɑ-syn pff on innate and adaptive immune responses, it is possible that different strains and aggregates of ɑ-syn might have differential effects. This is an area worth further investigation, given that in our control experiments with the monomeric ɑ-syn we detected subtle inflammatory effects in the amygdala. This is consistent with a recent study that showed differential effects of monomeric and aggregated ɑ-syn in the inflammasome in peripheral blood cells from PD patients [69]. Here it is worth pointing out that these as well as other recent studies were performed utilizing either ɑ-syn pff or ɑ-syn tg mice. Even though these models appear to show changes in T cells similar to patients with DLB/PD [36,38] additional confirmation will be needed in more physiological models not involving injection of exogenous ɑ-syn pff or overexpression of this protein. For example, studies in young vs aged ɑ-syn knock-in mice or the 3 K model developed by the group of Selkoe et al. [70] a tetramer abrogating model that results in PD like phenotype might be part in future studies. Moreover, recent studies suggest that ɑ-syn might trigger neurodegeneration through nonamyloid mechanisms [71]. Loss of function might be of interest for explaining immune system dysregulation since previous studies have shown that in ɑ-syn knockout mice there are alterations in the maturation of T and B cells [72][73][74]. Future studies will need to further explore these alternative mechanisms.
The mechanisms through which aging might contribute to the more severe T cell infiltration, microglialinflammatory responses and ɑ-syn accumulation are not completely understood. However, our transcriptomic and pathway analysis showing that in the young mice ɑ-syn pff triggered inflammatory networks similar to those observed in microglia from aged mice suggests complementary effects of aging and ɑ-syn aggregates. In fact, the age-associated increase in chronic, low-grade sterile inflammation has been termed "inflammaging" [11] and is strongly associated with age-associated disorders including AD, DLB, PD and other neurodegenerative disorders. Moreover, during aging, there is senescence of T cell populations. This includes reduced production of naïve T cells and accumulation of highly differentiated T cells resulting in a diminished T cell receptor repertoire [75]. We also observed similar activation of microglia during aging and upon exposure to the ɑ-syn aggregates that included increased size, decreased ramification and increased production of proinflammatory cytokines. This might be explained by an effect of ɑ-syn aggregates on microglial cell senescence, similar to what previously shown in aging microglia [76]. Further studies in this model will be needed to investigate the effects of ɑ-syn aggregates on T cell and microglial cell senescence.
In agreement with studies showing T cell infiltration following ɑ-syn pff in rodent models [42,60], recent studies have shown that in DLB [36,38] and PD [77,78] there is abundant T cell infiltration in the brain. Likewise, extensive T cell infiltration in the CNS accompanied by microglial activation have been shown in studies in ɑ-syn over-expressing mice mimicking DLB/PD [36,38] and multiple system atrophy [79], as well as neurotoxic models (MPTP challenge) in mice [80] and nonhuman primates [81]. Interestingly, alternative models where ɑ-syn pff was administered systemically [22] or via intra-enteric targeting of the ganglionic plexus have also shown dysregulation of immune responses with trafficking of lymphocytes, monocytes and macrophages into the CNS. Furthermore, depleting selected T-cell populations ameliorates dopaminergic neurodegeneration in animal models of PD, thus indicating a key role of adaptive immunity in the neurodegenerative process [57].
The molecular mechanisms through which agerelated alterations in innate immune responses might be involved in synucleinopathies has recently gained interest [24,77]. For example, accumulation and transmission of ɑ-syn to microglia leads to IL6 production and triggers alterations in neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export [82]. Here based on the transcriptomics analysis projected from IPA, we found a network that included novel regulators such as CSF2 and poly rl:rC-RNA as well as LPS/TLR and TNFɑ as common upstream transcriptional regulators. Interestingly, these pathways have several commonalities, including involvement in cytokine-driven neuroinflammatory process, signaling via toll-like receptors (TLRs) and regulation of microglial function. Notably, an important driver role of CSF2 (also known as granulocyte macrophage stimulating factor [GM-CSF]), which is a cytokine that controls the production, differentiation, and function of granulocytes and macrophages in the CNS, promotes microglia activation and inflammation [83]. Interestingly, CSF2 is produced by natural killer (NK) T cells [84] which remarkably traffic in increased numbers into the CNS of DLB/PD patients and ɑ-syn models [36], mediating neuroinflammation [85]. Microglia, among other cells in the CNS, express the CSF2R [86] that includes subunits A and B [87]; these subunits form complexes that signals via JAK2 to trigger neuroinflammation. In individuals with a rare kind of dementia with demyelination known as adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP); CSF2 is increased and colony stimulating factor 1 receptor (CSF-1R) is haploinsufficient [88]. Remarkably, a recent study showed that downregulation of CSF2 in CSF-1R deficient mice rescues the behavioral and neuropathological alterations in the mouse model of ALSP indicating that CSF2 is critical in regulating microglial function and inflammation in the CNS and has been proposed as a therapeutical target for ALSP [89]. Until this study, the role of CSF2 in PD/DLB and other neurodegenerative disorders had not been considered; however, increased levels of CSF-R1 have been described in AD and inhibitors of CSF-R1 have been proposed as a therapy for PD and other neurodegenerative disorders [90]. The implication of these studies is that in DLB/PD, CSF2 and other cytokines released by NK and T cells that traffic into the CNS activate microglia via CSFRs and thus CSF2R might be an important target to consider (Fig. 9). While the role of CSF2R is less well known, in recent years the role of TLRs in DLB/PD has been extensively considered [91] and TLR2 has been shown to be a key mediator of the neurotoxic and proinflammatory effects of extracellular ɑ-syn aggregates [26]. Taken together our network analysis showed that pathways involving stimulation of microglia via TLRs, and CSF-R are critical toward understanding the role of aging in DLB/PD (Fig. 9).
In conclusion the present study showed that aging triggered a more inflamed micro-environment in the ɑ-syn pff model of DLB/PD, resulting in increased T cell infiltration and microgliosis. Microglia displayed distinctive inflammatory transcriptomic patterns overlapping with genes differentially expressed in microglia in the aged mice. These results point to the possibility that targeting selected neuro-immune responses involving CSFR and TLRs might be important in developing treatments for DLB/PD.

Conclusions
Here we demonstrated that ɑ-syn pff injection in aged mice induced more spreading of ɑ-syn pathology in selected brain regions (striatum and amygdala), greater infiltration of T cells and microgliosis, and induced stronger behavioral deficits compared to young mice with ɑ-syn pff injection. Transcriptomic analysis of microglia revealed that ɑ-syn pff injected young mice had hallmarks of aged microglia. Thus, the enhanced ageassociated pathologies found in aged mice may have originated from inflammatory synergy between aging and the p-ɑ-syn aggregation. Furthermore, with ingenuity pathway analysis, we found a novel network of CSF2 and poly rl:rC-RNA as well as other previously known network of LPS/TLR or TNFɑ. We propose that common molecular mechanisms of immune dysregulation involving novel (CSF2, poly rl:rC-RNA) and other known (LPS/TLR, TNFɑ) networks converge in aging and ɑ-syn inflammatory neuropathogenesis. We suggest that targeting neuro-immune responses might be important in developing treatments for DLB/PD.